Processes for the growth of a modified Pasteurella multocida bacteria and preparation of a vaccine therefrom

ABSTRACT

The chemical modification of virulent Pasteurella multocida and Pasteurella haemolytica strains and preparation of live bacteria vaccines from the modified organisms for immunization of bovine, porcine and ovine animal species are disclosed.

This is a division of application Ser. No. 135,828 filed Mar. 31, 1980,now U.S. Pat. No. 4,293,545.

This invention relates to Pasteurella bacteria and the preparation ofvaccines therefrom. In particular, the invention relates to themodification of virulent Pasteurella multocida and Pasteurellahaemolytica strains, to the preparation of mono- and polyvalent livebacteria vaccines from the modified organisms and to processes forpreparing and using such vaccines.

Pasteurella multocida and Pasteurella haemolytica are known to infectbovine, porcine and ovine animal species causing respiratory disease,and have been implicated in the etiology of "shipping fever" syndrome.[Jensen et al., "Diseases of Feedlot Cattle", 3rd ed., Lea & Febiger,Philadelphia (1979), pgs. 59-65]. At present, pasteurellosis of domesticanimals is controlled, with varying degrees of success, by theadministration of vaccines, antimicrobial agents or a combination of thetwo.

Known vaccines for pasteurellosis contain killed whole cells(bacterins), live attenuated bacteria or cell fractions, with or withoutan adjuvant. One problem with the testing and use of such vaccines isthat the highly variable cross-protection among Pasteurella multocidaserotypes often results in the vaccine strain employed conferring littleor no immunity against an infecting organism encountered under fieldconditions [Collins, "Mechanisms of acquired resistance to Pasteurellamultocida infection: A review.", Cornell Vet. 67(1):103 (1977)].

Commercial bacterins, which usually contain one or more strains offormalin-killed Pasteurella, are undesirable due to several factors. Atleast two doses of such a vaccine, given several days apart, arenecessary for effective protection (Collins, supra); they are often notsuccessful and have been noted to cause transient endotoxic shock[Larson et al., J. Am. Vet. Med. Assn. 155:495 (1969)].

A number of combination vaccines containing killed Pasteurella are alsoknown and used. For example, killed Pasteurella has been combined foruse in cattle with bovine infections rhinotracheitis virus [Matsucka etal., J. Am. Vet. Med. Assn. 160(3):333 (1972)]; with bovineparainfluenza-3 virus [Sampson et al., Vet. Med. Small Anim. Clin.67(12):1354 (1972)]; U.S. Pat. No. 3,501,770 and U.S. Pat. No.3,526,696]; in quadrivalent form, wth bovine infectious rhinotracheitisvirus, bovine viral diarrhea mucosal disease virus and bovineparainfluenza-3 virus (U.S. Pat. No. 3,634,587); and with Salmonellatyphimurium (U.S. Pat. No. 4,167,560). German Offenlegungsschrift2,816,942 discloses a cellular vaccine for atrophic rhinitis in pigscomprising killed Pasteurella multocida and Bordetella bronchiseptica.Avian vaccines, particularly for use against fowl cholera, containingkilled Pasteurella alone or in combination with other bacteria orviruses are also known.

Avian and bovine vaccines containing live Pasteurella have also beendeveloped. A turkey vaccine containing live attenuated Pasteurellamultocida is described by Bierer et al., Poultry Science 47(4):1258(1968), and in U.S. Pat. No. 3,855,408. Rice et al., Poultry Science55(4):1605 (1976), describe the vaccination of chickens with a livePasteurella multocida vaccine and U.S. Pat. No. 4,169,886 discloses alive fowl cholera vaccine prepared from the M-3-G strain of Pasteurellamultocida.

Recently, a bovine vaccine for shipping fever, administered byintradermal injection, comprising a field strain live culture ofPasteurella haemolytica in a brain-heart infusion broth was disclosed inU.S. Pat. No. 4,171,354. Carter et al., Am. J. Vet. Res. 39(9):1534(1978) and 40(3):449 (1979), have developed a hemorrhagic septicemiavaccine using a live streptomycin-dependent mutant of Pasteurellamultocida which is highly immunogenic in mice, rabbits and calves. Thisvaccine also protected turkeys against fowl cholera [Chengappa et al.,Avian Disease 23(1):57 (1979)].

Immunization of turkeys, chickens and/or mice with various cellfractions of Pasteurella multocida has also been achieved, for examplewith culture filtrate, cell walls and cytoplasm [Brown et al., Appl.Microbiol. 19(5):837 (1970)], free endotoxin [Rebers et al., Am. J. Vet.Res. 35(4):555 (1974) and Canadian Pat. No. 1,030,873], aprotein-polysaccharide complex isolated from the bacteria [Ganfield etal., Infect. Immun. 14(4):990 (1976)], cell membrane [Borisenkova etal., Veterinariya (Mosc.) 5:48 (1977)], a glycoprotein extract of aculture filtrate [Srivastava et al., Can. J. Microbiol. 23(2):197(1977)] and a ribosomal fraction [Baba, Infect. Immun. 15(1):1 (1977)].Nagy et al., Res. Vet. Sci. 20(3):249 (1976), describe the protection ofcattle against hemorrhagic septicemia caused by Pasteurella multocidatype E organisms with a vaccine comprising an extract of Pasteurellacapsular material in an aluminum hydroxide adjuvant (see alsoNetherlands Pat. No. 73 04 320).

Ribosomal vaccines prepared from various organisms, includingPasteurella multocida and Pasteurella haemolytica, are described inBelgian Patent 857,014. A potassium thiocyanate extract of Pasteurellahaemolytica serotype 1 was found to be immunogenic and to producecross-immunity in mice against Pasteurella multocida type A [Mukkur,Infect. Immun. 18(3):583 (1977)]. Chickens and calves have beenprotected against heterologous challenge with a potassium thiocyanateextract of Pasteurella multocida serotype 3 [Gaunt et al., Avian Disease21(4):543 (1977); Mukkur, Am. J. Vet. Res. 39(8):1269 (1978)].

Until the present work, chemical modification of Pasteurella bacteriaand preparation of a safe and highly effective vaccine for protectinganimals, especially economically important feed animals, against theravages of "shipping fever" and other Pasteurella associated diseasesare not believed to have been accomplished. The vaccines produced fromthe new modified Pasteurella organisms of this invention have also beenfound to be cross-protective against a variety of Pasteurella spp fieldisolates.

One aspect of the present invention consists of safe and effectivevaccines for the protection of bovine, porcine and ovine species ofanimals against upper respiratory disease associated with Pasteurellainfection, including that commonly known as "shipping fever". Modifiedlive Pasteurella multocida and Pasteurella haemolytica monovalentvaccines have been prepared for administration by the subcutaneous,intranasal or, preferably, intramuscular route. For administration tobovine and porcine species, such vaccines preferably contain from about1.0×10⁷ to about 1.0×10¹¹ CFU (colony forming units) per dose of themodified live Pasteurella multocida or the modified live Pasteurellahaemolytica organisms with a suitable carrier and/or stabilizer. Foradministration to ovine species, the vaccine preferably contains fromabout 1.0×10⁹ to about 1.0×10¹¹ CFU/dose of the modified Pasteurellamultocida organism or from about 1.0×10⁷ to about 1.0×10¹¹ CFU/dose ofthe modified Pasteurella haemolytica organism. The vaccines areadministered in one or two doses, preferably two, of from 2.0 ml to 5.0ml each, depending on the species and size of the animal beingvaccinated as well as the organism count.

A bivalent vaccine consisting of vaccinal amounts of the modified livePasteurella multocida and modified live Pasteurella haemolyticadescribed herein is also an object of this invention. For administrationto bovine and porcine species, such vaccine contains from about 1.0×10⁷to about 1.0×10¹¹ CFU/dose of each of the modified live Pasteurellastrains with a suitable carrier and/or stabilizer. For administration toovine species, the vaccine contains from about 1.0×10⁹ to about 1.0×10¹¹CFU/dose of the modified live Pasteurella multocida organisms and fromabout 1.0×10⁷ to about 1.0×10¹¹ CFU/dose of the modified livePasteurella haemolytica organisms. Such vaccine may be administered bythe subcutaneous, intranasal or, preferably, intramuscular route in oneor two doses, preferably two, of from 2.0 ml to 5.0 ml each.

Another aspect of the present invention consists of the new modifiedPasteurella multocida and Pasteurella haemolytica organisms. These weredeposited with the American Type Culture Collection in Rockville, Md. onMar. 5, 1980 and have been assigned accession numbers 31610 (modifiedPasteurella multocida) and 31612 (modified Pasteurella haemolytica). Theorganisms will be freely available on request upon issuance of thisapplication, or any foreign equivalent thereof, as a patent.

The Pasteurella bacteria used to prepare the vaccines of this inventionwere isolated from lung tissue of infected animals and identified bystandard identification methods. Both isolates were shown to be virulentby inoculation into mice and hamsters. Propagation of the bacteria wascarried out in a liquid medium consisting of tryptose broth supplementedwith thiamine (Difco Laboratories, Detroit, Mich.). Tryptose agar with5% sheep blood was employed as a medium to determine the colonialcharacteristics of each of the Pasteurella parent bacteria.

The parent Pasteurella strains are chemically modified with acridiniumsalts, such as 3,6-bis-dimethylamino acridinium chloride (acridineorange), 2,8(3,6)diamino-10-methyl acridinium chloride and2,8(3,6)diamino acridinium chloride (acriflavine HCl), in concentrationsof from about 0.1 μg/ml to about 150 μg/ml in a medium consisting oftryptose supplemented with thiamine broth. The organisms may be passagedup to about 40 times in acridinium salt-supplemented broth with fromabout 8 to about 26 passages being preferred for the Pasteurellamultocida and from about 10 to about 30 passages being preferred for thePasteurella haemolytica. Upon modification, the morphologicalcharacteristics of the bacteria change from smooth, glistening andmucoid colonies of 1.5-2.0 mm diameter after incubation at 37° for 18hours to rough, dull and punctiform colonies of 0.5-1.0 mm diameterafter incubation. The chemically modified bacteria may be further grownand passaged in any suitable growth media, for example in tryptose brothsupplemented with thiamine or in the medium described herein.

The vaccines of this invention are prepared by standard, known to theart methods, for example by combining the bacteria with a suitablecarrier and/or a stabilizer.

DETAILED DESCRIPTION OF THE INVENTION Isolation, Propagation andChemical Modification of the Pasteurella Multocida Vaccine Strain

The virulent Pasteurella multocida used to prepare the modified liveorganism of this invention (ATCC No. 31609) was obtained from andidentified by the University of Nebraska, Department of VeterinarySciences and was originally isolated from lung tissue of a gnotobioticcalf infected with the bacteria. The calf had been inoculated by theintratracheal route with a suspension of a pool of lung tissues from twodiseased calves which had previously been similarly inoculated withpulmonary materials obtained from samples presented for laboratorystudies. Within 36 hours following inoculation, the calf manifestedclinical symptoms of a bacterial pneumonia, viz. elevated temperature,depression, cough and labored breathing. The animal was euthanized and,at necropsy, severe pneumonia with a fibrinous pleuritis was noted.Samples of the lung tissues were obtained aseptically and frozen at -50°C.

Samples of the frozen tissues were thawed and streaked on blood agarplates. Selected colonies of the growth were identified by standardbiochemical reactions as being typical of Pasteurella multocida. Otherselected colonies were inoculated into a medium or tryptose brothsupplemented with thiamine. Growth of the organisms was allowed toproceed at 37° C. for 16 hours. Aliquotes of the culture were thendispensed into small sterile vials which were stoppered with sterileneoprene stoppers, sealed and stored at -70° C.

A vial of the frozen broth culture was thawed and inoculated into a 100ml broth culture of tryptose broth supplemented with thiamine. Followingincubation at 37° C. for 21 hours, a 1.0 ml volume of the culture wasinoculated by the intracardiac route into a young adult New Zealandwhite rabbit. The inoculum contained about 2.0×10⁷ CFU/ml. The rabbitwas sacrificed eight hours after inoculation. Samples of liver andspleen and a quantity of blood were obtained. The tissues werehomogenized, combined with the blood and frozen at -70° C.

A vial of the frozen rabbit tissue containing the Pasteurella multocidaorganisms was thawed and two passages of the virulent organism were madein tryptose broth supplemented with thiamine. A small amount of thesecond passage material was inoculated into tryptose broth supplementedwith acriflavine HCl at a level of 0.75 μg/ml. Following incubation for24 hours at 37° C., a small amount of the bacterial growth wasinoculated into tryptose broth containing 1.5 μg/ml of acriflavine HCl.Additional passages were made in tryptose broth supplemented with 1.5μg/ml of acriflavine HCl.

Each passage of the organism in the presence of acriflavine HCl wasmonitored by streaking out the growth on the surface of blood agarplates to observe the purity of the culture and any changes in themorphology of the organism colonies.

A change in the morphology of the colonies of the modified Pasteurellamultocida strain was noted after a total of eight passages in thepresence of acriflavine HCl. The parent organism and the organisms ofthe early passages in the presence of acriflavine HCl were smooth,glistening and mucoid. The size of these colonies following incubationat 37° C. for 18 hours was from 1.5 to 2.0 mm in diameter. The coloniesof the eighth passage organisms streaked out on blood agar plates wererough and punctate. The size of the colonies was between 0.5 to 1.0 mmfollowing incubation at 37° C. for 18 hours.

The chemically modified Pasteurella multocida strain was furtherpassaged in acriflavine HCl supplemented broth and tested for purity andanimal (hamster and mouse) LD₅₀ values following the 8th, 15th, 20th,26th and 30th passages. The test animals were administered 0.1 ml of thevaccinal strain containing approximately 1.0×10⁶⁻⁸ CFU by theintraperitoneal route. Following vaccination, the animals werechallenged with known-virulent strains of Pasteurella multocida. Thechallenge strains employed were the Carter B (bison) strain, USDA strain#169 and USDA strain #1062 or isolates of Pasteurella multocida obtainedfrom various university diagnostic laboratories. The challenge organismsgenerally had relatively low LD₅₀ values of from 1 to 100 organisms.

Those animals which had previously been vaccinated with one or two dosesof the chemically modified strain from the 8th to the 26th passagelevels resisted challenges of from one to greater than 1.0×10⁷ virulentorganisms. At the 26th passage level, the vaccine strain protected allof the vaccinated animals.

A single small colony of the modified Pasteurella multocida organism,26th passage, was isolated and inoculated into tryptose brothsupplemented with thiamine. Following incubation at 37° C. for 18 hours,a stabilizer was added to the growth medium as a freezing menstruum. Theorganism was dispensed in a number of vials which were frozen at -70° C.This lyophillized organism was deposited with the American Type CultureCollection in Rockville, Md. on Mar. 5, 1980 and has been assignedaccession number 31610.

To determine the genetic stability of the modified Pasteurella multocidaof this invention (ATCC No. 31610), the 26th passage material waspassaged an additional 15 times in tryptose broth supplemented withthiamine. At the 5th, 10th and 15th passage levels in the absence ofacriflavine, morphology of the colonies on blood agar plates was similarto that following exposure to 26 passages in acriflavine-containingmedium. The protective properties of the 5th, 10th and 15th passages inacriflavine-free broth material remained unchanged. Virulence of theorganisms after the 5th, 10th and 15th passages in acriflavine-freebroth remained low and was equal to or greater than 1.0×10⁷ organisms.

Preparation and Use of the Modified Live Pasteurella Multocida Vaccine

For vaccine preparation, the modified Pasteurella multocida strain (26thpassage material) is further propagated in a suitable growth medium. Anexample of such suitable medium follows:

    ______________________________________                                        Ingredient     Grams/Liter of Water                                           ______________________________________                                        Bacto-Peptone  10.0-40.0                                                      HY-Case Amino  5.0-20.0                                                       NZ-Amine A     5.0-20.0                                                       NZ-Amine B     5.0-20.0                                                       Bacto-Yeast Extract                                                                          5.0-20.0                                                       Sodium Chloride                                                                              0.5-3.0                                                        ______________________________________                                    

The above ingredients are combined and sterilized by autoclaving. Asolution of 20.0-80.0 grams/liter of sucrose is separately sterilized byautoclaving and added to the other ingredients when cooled. The pH isadjusted to 7.4-7.6 with 10 N sodium hydroxide solution.

From one to four parts of growth medium containing the modifiedorganisms are combined with one part of a stabilizer and lyophilized. Anexample of a suitable stabilizer follows:

    ______________________________________                                        Solution 1                                                                                           Grams/Liter                                            Ingredients            of Water                                               ______________________________________                                        Potassium Hydroxide (anhydrous)                                                                      0.2-0.8                                                L-glutamic acid        0.5-2.0                                                Potassium phosphate dibasic (anhydrous)                                                              1.0-4.0                                                Potassium phosphate monobasic (anhydrous)                                                            0.3-1.5                                                Sucrose                 50-200                                                ______________________________________                                    

The ingredients are combined and sterilized by autoclaving.

    ______________________________________                                        Solution 2                                                                    Ingredients          Grams/Liter of Water                                     ______________________________________                                        Gelatin (Knox)       100-300                                                  Autoclave for four hours to hydrolyze.                                        ______________________________________                                    

Two parts of Solution 2 are added to three parts of Solution 1 toprepare the stabilizer solution.

VACCINATION OF CALVES

The modified live Pasteurella multocida vaccine of this invention wasadministered in two 5.0 ml doses given by the subcutaneous orintramuscular routes at two week intervals to ten calves found to bedevoid of protective antibodies. Nine of the calves were conventionaldairy calves which had been deprived of colostrum after birth and onewas a gnotobiotic animal obtained by cesarean section and maintained inan isolation unit. Two weeks following administration of the second doseof vaccine, all of the calves were challenged by intratrachealadministration of Pasteurella multocida Carter type B organisms ofdemonstrated virulence when administered to calves by the subcutaneousand intratracheal routes. The results of this test appear in Table 1.

                  TABLE I                                                         ______________________________________                                        Protection Afforded Calves by Vaccination with a Modified Live                Vaccine Prepared from ATCC No. 31610 Against a Pasteurella                    Multocida Carter type B Challenge                                             Vaccination                                                                   (CFU/5.0 ml)                                                                        First    Second            Status                                       Animal                                                                              Dose     Dose     Route    Post-Challenge                               ______________________________________                                        1*    2.5 × 10.sup.9                                                                   4.5 × 10.sup.8                                                                   Subcutaneous                                                                           Dead                                                                          (120 hrs. P.C.)**                            2     3.4 × 10.sup.9                                                                   5.5 × 10.sup.8                                                                   Subcutaneous                                                                           Normal                                       3     3.4 × 10.sup.9                                                                   5.5 × 10.sup.8                                                                   Subcutaneous                                                                           Normal                                       4     3.4 × 10.sup.9                                                                   5.5 × 10.sup.8                                                                   Subcutaneous                                                                           Normal                                       5     3.4 × 10.sup.9                                                                   5.5 × 10.sup.8                                                                   Subcutaneous                                                                           Normal                                       6     3.4 × 10.sup.9                                                                   5.5 × 10.sup.8                                                                   Intramuscular                                                                          Normal                                       7     3.4 × 10.sup.9                                                                   5.5 × 10.sup.8                                                                   Intramuscular                                                                          Normal                                       8     3.4 × 10.sup.9                                                                   5.5 × 10.sup.8                                                                   Intramuscular                                                                          Normal                                       9     3.4 × 10.sup.9                                                                   5.5 × 10.sup.8                                                                   Intramuscular                                                                          Normal                                       10    3.4 × 10.sup.9                                                                   5.5 × 10.sup.8                                                                   Intramuscular                                                                          Normal                                       A+    --       --       --       Dead (48 hrs. P.C.)                          B+    --       --       --       moribund, sacrificed                         ______________________________________                                         *Gnotobiotic?                                                                 **no Pasteurella found at necropsy                                            +Control                                                                 

VACCINATION OF SWINE

The modified live Pasteurella multocida vaccine of this invention wasadministered to four normal feeder weight pigs of from about 30-50pounds each which were determined to be free of protective antibodies toPasteurella multocida USDA strain #169. The animals were vaccinated withtwo 5.0 ml doses administered intramuscularly 20 days apart. Two weeksfollowing the administration of the second dose of the vaccine, thevaccinated animals and five non-vaccinated, serologically negativecontrol animals were challenged by intravenous inoculation of 1.5×10⁹organisms of Pasteurella multocida USDA strain #169 contained in a 5.0ml volume.

The vaccinated and control animals were maintained in separate cleanrooms in an isolation building following challenge and were observed atleast twice daily for 14 days. About three hours following theadministration of the challenge material, the control animals showedsigns of depression and respiratory distress. All of the control animalsbecame uncoordinated and at about eight hours following the challengethey were recumbent. Two of the control animals died of an acutepneumonia at 40 and 58 hours following challenge. The remaining threecontrol animals showed signs of respiratory difficulties and wereappreciably depressed for several days. One of the control animalsreturned to a normal condition by the end of the observation periodwhile the other two control animals remained depressed and failed torecover fully from the effects of the challenge.

The vaccinated animals were depressed and off feed for the first 8 to 12hours following challenge, but returned to a normal state within 24hours.

The results of this test appear in Table II.

                  TABLE II                                                        ______________________________________                                        Protection Afforded Swine by Vaccination with a Modified Live                 Vaccine Prepared from ATCC No. 31610 Against Pasteurella                      Multocida USDA Strain #169 Challenge                                          Vaccination (CFU/5.0 ml)                                                                         Status                                                     Animal                                                                              First Dose                                                                              Second Dose                                                                              Post-Challenge                                                                          Comments                                 ______________________________________                                        1     9.0 × 10.sup.9                                                                    1 × 10.sup.10                                                                      Alive and Well                                                                          Normal                                   2     9.0 × 10.sup.9                                                                    1 × 10.sup.10                                                                      Alive and Well                                                                          Normal                                   3     9.0 × 10.sup.9                                                                    1 × 10.sup.10                                                                      Alive and Well                                                                          Normal                                   4     9.0 × 10.sup.9                                                                    1 × 10.sup.10                                                                      Alive and Well                                                                          Normal                                   A*    --        --         Poor doing                                                                              Unthrifty                                B*    --        --         Poor doing                                                                              Returned                                                                      to Normal                                C*    --        --         Died                                                                          (58 hrs P.C.)                                      D*    --        --         Poor doing                                                                              Unthrifty                                E*    --        --         Died                                                                          (40 hrs P.C.)                                      ______________________________________                                         *Control                                                                 

VACCINATION OF SHEEP

The modified live Pasteurella multocida vaccine of this invention wasadministered to ten unvaccinated susceptible sheep ranging in weightfrom 40 to 130 pounds which were obtained from a flock having a historyof being free of respiratory disease problems. The animals were dividedinto two groups which were vaccinated with two different levels ofvaccine. One group of animals received vaccine containing about 1.0×10¹⁰CFU/5.0 ml dose and the other group was vaccinated with approximately4.0×10⁸ CFU/5.0 ml dose. The interval of time between the administrationof the two doses of the vaccine was 20 days.

Fourteen days following the second vaccination the vaccinated sheep anda control group were challenged by intravenous administration ofapproximately 3.0×10⁹ CFU/3.0 ml volume of Pasteurella multocida USDAstrain #1062. Following challenge, the animals were observed at leasttwice a day for a period of 14 days.

It was noted that within four hours after challenge the control animalsbecame depressed and exhibited signs of respiratory distress. Withinseveral hours the animals of this group became increasingly moredepressed and breathing became labored. Two of the control animals diedof pneumonia, one at 38 hours following challenge and one at 148 hours.The surviving three animals remained depressed and were recumbent forprolonged periods. Each of the control animals develop joint swellingsand exhibited depression and varying degrees of locomotion and breathingdifficulty.

The five sheep vaccinated with two reduced doses of the vaccine showedessentially the same signs and symptoms following challenge as did thecontrol animals. Three of these animals died at 65, 73 and 294 hoursfollowing challenge. Extensive penumonia was observed in each of theseanimals at necropsy. The two surviving animals failed to return to anormal status and were weak and depressed throughout the post-challengeperiod.

The animals vaccinated with two doses of vaccine containing 1.0×10¹⁰CFU/dose were slightly depressed and went off feed immediately followingchallenge. Within 24 hours after challenge the animals, with theexception of one animal which developed a limp, returned to normal.During the remainder of the observation period, the animals of thisgroup exhibited transient lameness and periodically showed somedepression. One animal became depressed and quite lame near the end ofthe observation period and was sacrificed at termination of the test. Atnecrospy a small area of pneumonia was observed in one lobe andPasteurella multocida-like organisms were recovered from the involvedjoint fluids.

Table III presents the results of this test.

                  TABLE III                                                       ______________________________________                                        Protection Afforded Sheep by Vaccination with a Modified Live                 Vaccine Prepared from ATCC No. 31610 Against a Pasteurella                    Meltocida Strain #1062 Challenge                                              Vaccination                                                                   (CFU/5.0 ml)                                                                        First    Second   Status                                                Animal                                                                              Dose     Dose     Post-Challenge                                                                          Comments                                    ______________________________________                                        1     9.0 × 10.sup.9                                                                   1.0 × 10.sup.10                                                                  Alive     Normal                                      2     9.0 × 10.sup.9                                                                   1.0 × 10.sup.10                                                                  Alive     Normal                                      3     9.0 × 10.sup.9                                                                   1.0 × 10.sup.10                                                                  Alive     Normal                                      4     9.0 × 10.sup.9                                                                   1.0 × 10.sup.10                                                                  Alive     Depressed,                                                                    Lameness,                                                                     Sacrificed                                  5     9.0 × 10.sup.9                                                                   1.0 × 10.sup.10                                                                  Alive     Normal                                      6     3.6 × 10.sup.8                                                                   4.0 × 10.sup.8                                                                   Poor doing                                                                              Sacrificed                                  7     3.6 × 10.sup.8                                                                   4.0 × 10.sup.8                                                                   Poor doing                                                                              Died (32 days                                                                 P.C.)                                       8     3.6 × 10.sup.8                                                                   4.0 × 10.sup.8                                                                   Died                                                                          (254 hrs P.C.)                                        9     3.6 × 10.sup.8                                                                   4.0 × 10.sup.8                                                                   Died                                                                          (73 hrs P.C.)                                         10    3.6 × 10.sup.8                                                                   4.0 × 10.sup.8                                                                   Died                                                                          (65 hrs P.C.)                                         A*    --       --       Poor doing                                                                              Depressed, Joint                                                              Problems                                    B*    --       --       Poor doing                                                                              Depressed, Joint                                                              Problems                                    C*    --       --       Poor doing                                                                              Died (25 days                                                                 P.C.)                                       D*    --       --       Died                                                                          (38 hrs P.C.)                                         E*    --       --       Died                                                                          (148 hrs P.C.)                                        ______________________________________                                         *Control?                                                                

Isolation, Propagation and Chemical Modification of the PasteurellaHaemolytica Vaccine Strain

The parent Pasteurella haemolytica used to prepare the modified liveorganism of this invention (ATCC No. 31611) was isolated from lungtissue aseptically removed from a calf, submitted to the University ofNebraska, Department of Veterinary Sciences, which had died from"shipping fever" and frozen at -50° C.

Samples of the frozen tissues were thawed and streaked on the surfacesof sheep blood agar plates. Following incubation at 37° C. for 24 hours,a pure culture of colonies resembling Pasteurella spp was observed.Several colonies were selected and inoculated into test media foridentification. The results of the tests indicated the organism to bePasteurella haemolytica.

Other colonies of the organism were inoculated into a medium of tryptosebroth supplemented with thiamine. Following incubation at 37° C. for 24hours, an additional passage was made in the same medium. A volume of astabilizer solution, described above, was added to the growth of theorganism. The growth-stabilizer mixture was dispensed in 2.0 ml aliquotsand subjected to lyophilization. The lyophilized parent organism wasstored at 4° C. Colonies of the parent organism following incubation at37° C. for 24 hours were circular, glistening and mucoid. The colonieswere about 2.0 mm in diameter.

A lyophilized sample of the parent organism was rehydrated, inoculatedinto a medium of tryptose broth supplemented with 1.5 μg of acriflavineHCl/ml and was subjected to three passages in this broth. In subsequentpassages, the concentration of acriflaving HCl was increased. In the 6thpassage, the concentration of acriflavine HCl was 15.0 μg/ml. In the10th passage, the colonies of the organism were punctiform, dull andrough with a diameter of about 0.5 mm.

Concomitant with the changes in the size and characteristics of thecolonies of the acriflavine-treated organisms, a marked reduction in thevirulence of the modified organisms were noted. The LD₅₀ values in micefor the parent and chemically modified strains were 2.9×10³ and 7.3×10⁶CFU, respectively. In hamsters, the LD₅₀ values for the parent organismand for the chemically modified strain were 1.1×10⁶ and ≧6.2×10⁸ CFU,respectively.

Following 12 passages in acriflavine HCl-supplemented medium, themodified organism was passaged 15 times in a medium not supplementedwith acriflavine HCl. The 15th passage level material produced LD₅₀values in mice and hamsters nearly identical to that produced bymaterial prior to passage in acriflavine-free medium.

Preparation and Use of the Modified Live Pasteurella Haemolytica Vaccine

For preparation of a vaccine, further quantities of the modifiedPasteurella haemolytica (12th passage) are grown in a suitable medium,such as that described above for propagation of the modified Pasteurellamultocida, combined with a stabilizer and lyophilized. An example of astabilizer which may be employed is described above.

A further aspect of this invention is the preparation and use of acombination vaccine consisting of vaccinal amounts of the modified livePasteurella multocida and the modified live Pasteurella haemolyticabacteria. Such combination vaccine will, preferably, contain from about1.0×10⁷ to about 1.0×10¹¹ CFU/dose of each of the modified livePasteurella strains for vaccination of bovine and porcine species orfrom about 1.0×10⁹ to about 1.0×11¹¹ CFU/dose of the modified livePasteurella multocida strain and from about 1.0×10⁷ to about 1.0×10¹¹CFU/dose of the modified live Pasteurella haemolytica strain forvaccination of ovines. The vaccine can be prepared for subcutaneous,intramuscular or intranasal administration and is administered in one ortwo doses of from 2.0 ml to 5.0 ml each.

The preparation and use of such combination vaccines is carried outaccording to procedures described herein or within the knowledge ofthose skilled in the art of vaccine production and use.

What is claimed is:
 1. A process for preparing a modified livePasteurella multocida vaccine capable of inducing immunity in bovine,porcine and ovine animal species without serious side effects whichcomprises chemically modifying virulent Pasteurella multocida strainATCC No. 31609 by passaging it in the presence of an acridinium salt andcombining the modified bacteria with a carrier.
 2. The process of claim1 wherein the virulent organism is passaged from about one to about 40times in the presence of an acridinium salt.
 3. The process of claim 2wherein the virulent organism is passaged from about 8 to about 26 timesin the presence of an acridinium salt.
 4. The process of claim 3 whereinthe acridinium salt is acriflavine HCl.
 5. The process of claim 4wherein the virulent organism is passaged 26 times.
 6. The process ofclaim 5 wherein the modified Pasteurella multocida bacteria is ATCC No.31610.
 7. A process for preparing a further quantity of the modifiedPasteurella multocida bacteria ATCC No. 31610 which comprises growingsaid bacteria in a suitable growth medium for a length of timesufficient to permit growth of a greater amount of said bacteria.
 8. Theprocess of claim 7, wherein the growth medium is tryptose brothsupplemented with thiamine.
 9. The process of claim 7, wherein thegrowth medium is of the composition:

    ______________________________________                                        Ingredient     Grams/Liter of Water                                           ______________________________________                                        Bacto-Peptone  10.0-40.0                                                      HY-Case Amino  5.0-20.0                                                       NZ-Amine A     5.0-20.0                                                       NZ-Amine B     5.0-20.0                                                       Bacto-Yeast Extract                                                                          5.0-20.0                                                       Sodium Chloride                                                                              0.5-3.0                                                        ______________________________________                                    


10. The process of claim 7, 8 or 9 wherein the growth medium contains astabilizer.